REVIEW published: 16 March 2016 doi: 10.3389/fevo.2016.00017

Phylogeny and Classification of Euglenophyceae: A Brief Review

Carlos E. de M. Bicudo 1* and Mariângela Menezes 2

1 Department of Ecology, Instituto de Botânica, São Paulo, Brazil, 2 Phycology Laboratory, Botany, Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Euglenophyceae (phototrophic ) are an important lineage within the Euglenida, . Most of the approximately 3000 described species are free-living, phototrophic, unicellular flagellates with one to several plastids of secondary origin, three bounding membranes and chlorophylls a and b; but, the lineage also includes colorless species that lost their photosynthesizing capability. They show a typical cell membrane consisting of parallel proteinaceous strips and microtubules located underneath the plasma membrane, and discoid mitochondrial cristae. Euglenozoa are a monophyletic group that includes, besides Euglenida, the Kinetoplastidea, Diplonemea, and Symbiontida. Since the class Euglenophyceae was proposed, its classification system has undergone several revisions, mainly after the adoption of molecular techniques. This article summarizes recent advances in the phylogeny and classification of the phototrophic euglenids, aiming at understanding the situation of the group within the phylum Euglenozoa, as well as its evolutionary relationships and the changes in its

Edited by: taxonomic classification. The current status of the group, as well as the limitations derived Jan Maria Kwiatowski, from the lack of inclusion of tropical strains in phylogenetic studies is briefly discussed. University of Warsaw, Poland Keywords: Euglenophyceae, molecular biology, morphology, phylogeny, Reviewed by: Alberto G. Sáez, Instituto de Salud Carlos III, Spain Anna Karnkowska, INTRODUCTION University of British Columbia, Canada *Correspondence: The class Euglenophyceae includes phototrophic members of the Euglenida, supergroup , Carlos E. de M. Bicudo being a frequent component of the plankton in marine, brackish, and freshwater environments. [email protected] The class encompasses free-living phototrophic unicellular flagellates, with one to several plastids of secondary origin, with three bounding membranes and chlorophylls a and b; but, it also Specialty section: includes colorless species that lost their capability to photosynthesize as well as the photosensory This article was submitted to apparatus associated with cilia (secondary osmotrophic species). The Euglenida (euglenids), Phylogenetics, Phylogenomics, and together with Kinetoplastea (trypanosomes), Diplonemea (genera and Rhynchopus), Systematics, and Symbiontida, form the phylum Euglenozoa (Cavalier-Smith, 1981, 1993; Yubuki et al., 2009; a section of the journal Breglia et al., 2010). The phylum Euglenozoa is well-supported in molecular analyses. The most Frontiers in Ecology and Evolution prominent common features of this clade are the polycistronic transcription, massive trans- Received: 11 February 2015 splicing, and more rarely the absence of introns (Flegontov and Lukeš, 2012). The members Accepted: 16 February 2016 of Kinetoplastea are free-living and obligatory parasitic protists that have cells with extensive Published: 16 March 2016 mitochondrial DNA, termed kinetoplast or DNA kinetoplast; whereas Diplonemea or diplonemids Citation: may be free-living phagotrophs or facultative parasites, with sack-shaped cells and short, thin Bicudo CEdM and Menezes M (2016) flagella (Roy et al., 2007; Adl et al., 2012). The Symbiontida includes organisms living in low-oxygen Phylogeny and Classification of Euglenophyceae: A Brief Review. sediments; their cells possess rod-shaped epibiotic bacteria (Breglia et al., 2007; Adl et al., 2012). All Front. Ecol. Evol. 4:17. these groups share morphological features including paraxial rods, a regular array of longitudinal doi: 10.3389/fevo.2016.00017 subpellicular microtubules, and closed mitosis (Simpson, 1997).

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The advent of molecular studies and the increasing of the which exhibit a single highly branched mitochondrion, with a few number of genes and DNA regions used in the phylogenetic flat, lamellar cristae, and mitochondrial DNA arranged in circular studies led to a restructuring of the taxonomy of Euglenophyceae, chromosomes of two slightly different sizes (Marande et al., 2005; improved understanding of the phylogenetic positions of the Roy et al., 2007). genera and several species, and helped to deduce the phylogenetic The Symbiontida is a more recently described clade, which relationships within the group. shares typical characters with the Euglenozoa and also shows The present article summarizes the main information on features not previously described for this group, such as modified the phylogenetic position and taxonomic classification of the (hydrogenosome-like) mitochondria, an “extrusomal pocket,” phototrophic euglenids. The current status and limitations the highly organized extracellular matrix underneath epibiotic derived from the lack of inclusion of tropical strains in bacteria, and the complex flagellar transition zone (Yubuki et al., phylogenetic studies of this group are also discussed. A glossary 2009). of terms used in this review is included at the end of the The phylogenetic relationships among euglenids, article. diplonemids, kinetoplastids, and symbiontids remain controversial. Studies on morphological features (e.g., shape of the feeding and flagellar apparatuses, mitosis pattern, PHYLOGENETIC POSITION OF and cytoskeletal composition) suggest that euglenids and EUGLENOPHYCEAE WITHIN kinetoplastids are more closely related (Triemer and Farmer, EUGLENOZOA 1991). On the other hand, studies combining morphological and molecular phylogenetic data suggest that kinetoplastids and Euglenida, together with the Kinetoplastea, Diplonemea, and diplonemids are sister groups, with euglenids branching basally Symbiontida, form the phylum Euglenozoa. Cells with two cilia, to the first two groups (Simpson and Roger, 2004; Marande inserted apically or subapically in a pocket, characterize the and Burger, 2007; Kiethega et al., 2011). The discovery of the phylum Euglenozoa. The cells have a basic ciliary apparatus Symbiontida raised the question of whether diplonemids and pattern consisting of two functional kinetosomes and three symbiontids might be more closely related to euglenids than to asymmetrically arranged microtubular roots, with paddle- kinetoplastids (Yubuki et al., 2013). shaped, discoid or flat mitochondrial cristae, tubular extrusomes, A phylogenetic analysis based on SSU rDNA sequences and closed mitosis with an intranuclear spindle (Simpson, 1997; showed the Euglenophyceae as a robust monophyletic clade, Roy et al., 2007; Adl et al., 2012). with Eutreptia and Eutreptiella as the basal lineages and Euglenozoa are monophyletic (Breglia et al., 2010; Burki, Rapaza viridis (mixotrophic species) branching as sister lineage 2014) and their clades are well-supported by morphological (Yamaguchi et al., 2012; Figure 2). Euglenophyceae together and molecular data (Simpson, 1997; Simpson and Roger, 2004). with Rapaza form a well-supported clade sister to the clade Table 1 summarizes the main differences among Euglenida, encompassing heterotrophic groups—Anisonema, Peranema, Kinetoplastea, Diplonemea, and Symbiontida. Dinema, Distigma, Rhabdomonas (with good support), and The phylum Euglenozoa is a well-defined group, however, Peranema trichophorum is basal to this whole clade. Other its relationship to other protists remains uncertain. The cell heterotrophic euglenids (Entosiphon, Pleotia, Petalomonas, structure, especially the presence of discoid mitochondrial Notosolenus) including Symbiontida are grouped together cristae, and multigene phylogenetic analyses have suggested without good support. that the Euglenozoa are close to the class Heterolobosea, a group represented by heterotrophic amoebae, amoeboflagellates and flagellates (Simpson et al., 2006; Grant and Katz, 2014). CHANGES IN THE TAXONOMIC Information on ultrastructure and phylogeny suggests that both CLASSIFICATION OF EUGLENOPHYCEAE lineages are members of the supergroup Excavata (Hampl et al., 2009; Yabuki et al., 2011; Figure 1). Early Studies The Euglenida comprise a group of predominantly free- The euglenids are part of an extremely diverse lineage of living organisms characterized by a typical cell cover made flagellate protists (Marin et al., 2003). Ehrenberg (1830) of parallel proteinaceous strips and microtubules located first attempted to classify euglenids, when he described the underneath the plasma membrane, the pellicle; and encompasses genus Euglena. Ehrenberg (1830), keeping within his self- phototrophic, heterotrophic (osmotrophic and phagotrophic), created classification system, placed the new genus among and mixotrophic species (Leander et al., 2007; Yamaguchi et al., the Polygastrica of family Astasiae, i.e., multi-stomached 2012). creatures with no alimentary canal, variable body shape, but The Kinetoplastea includes parasitic species and free-living no pseudopods or lorica. Subsequently, several investigators bodonids, which have mitochondrial inclusions of distinctively added descriptive characteristics for the genus Euglena and arranged DNA molecules, termed a kinetoplast DNA or kDNA, established different classification systems for euglenids, based on which represents an apomorphic character of kinetoplastids the presence, number of flagella, and nutrition mode (Dujardin, (Simpson et al., 2004; Lukeš et al., 2009). 1841; Cohn, 1853; Klebs, 1883). The Diplonemea (diplonemids) encompasses a small group Hollande (1942) accomplished a major revision of these of mostly free-living phagotrophs and some facultative parasites, flagellates, grouping them by their shared structural features

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TABLE 1 | Main differences among Euglenida, Kinetoplastea, Diplonemea, and Symbiontida (numbers following some terms correspond to their definitions in the glossary at the end of the text).

Euglenida Kinetoplastea Diplonemea Symbiontidaaa

Feeding apparatus Integrated system of proteinaceous With cytostome8 that leads into a Distinct feeding apparatus with Feeding apparatus supported by “rods”34 and “vanes”42 Many deep cytopharynx6 supported by “vanes”42 a complex MRT21 phototrophic taxa with cytoplasmic (MRT)21 pockets reinforced by (MRT)21 Flagella With (PFR)29connected to the With (PFR)29connected to the In general without (PFR)29 Without flagellar hair flagellar axoneme flagellar axoneme mtDNA Numerous linear fragments of Kinetoplast (kDNA)14 Numerous small circular Arranged in circular different sizes molecules chromosomes of 2 slightly different sizes, distributed in a pankDNA-like fashion Mitochondria Discoid cristae Discoid cristae Highly branched, with few Mitochondrion derived organelles flattened cristae with reduced or absent cristae that lie beneath the plasma membrane Cytoskeleton7 Pellicle 28 Thick cell surface with glicocalyx Longitudinal bands of With strip-like S- shaped folds and submembrane microtubules microtubules lining the cell cortex and rod-shaped bacteria

FIGURE 1 | Unrooted phylogenetic tree showing the relationships among representatives of major lineages, based on a variety of molecular and morphological data. Green and red text indicates green and red algae derived lineages, respectively (Adapted from Katz, 2012).

(e.g., body shape, nutritional mode, flagellar structure, and Rhabdomonadales, Sphenomonadales, Heteronematales, degree of metaboly) into Peranemoidées, Petalomonadinées, and and Euglenamorphales); considered the reduction in the Euglenidinées. Hollande’s (1942) classification was accepted as flagella number as the most important evolutionary event for best reflecting the natural relationships among these organisms, understanding the phylogeny of the group; and separated the and consequently was widely adopted (Pringsheim, 1956; photosynthetic from the colorless forms, with a phagotrophic Leedale, 1967, 1978; Johnson, 1968). ancestor involving phototrophic forms through secondary The classification system of Leedale (1967, 1978) endosymbiosis (Leedale, 1967, 1978). Some taxa included in expanded Hollande’s (1942) scheme by incorporating new Leedale’s scheme underwent taxonomic changes based on physiological data and aspects of their ultrastructure. This analyses using molecular techniques. These taxa encompass classification established six orders (Eutreptiales, Euglenales, descendants that have independently lost their .

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FIGURE 2 | Maximum-likelihood tree inferred from SSU rDNA sequences showing the phylogenetic position of the Euglenophyce within Euglenozoa. Only support values above 50% for the ML bootstrap analysis are shown. Thick branches indicate Bayesian posterior probabilities (PP) over 0.95. The branch leading to the fast-evolving Entosiphon clade has been shortened by one half (indicated by “1/2”; Adapted from Yamaguchi et al., 2012).

The Leedale scheme of 1978 was used to classify the euglenids small subunit) sequences, the phylogenetic results for the group until 1986, when Euglena gracilis was sequenced using the SSU were merely preliminary, since the E. gracilis sequences were rRNA gene (Sogin et al., 1986). compared with only a small number of sequences from other (Sogin et al., 1986). A more-robust molecular study, Molecular Studies also based on SSU rDNA sequence data, for 10 species (6 Single-Gene Phylogenies phototrophs and 4 heterotrophs), suggested that euglenids are a Although E. gracilis was the first phototrophic to be distinct monophyletic clade, the phototrophic forms arose after studied molecularly, based on the SSU rDNA (ribosomal nuclear the phagotrophic members, the biflagellate form diverged prior to

Frontiers in Ecology and Evolution | www.frontiersin.org 4 March 2016 | Volume 4 | Article 17 Bicudo and Menezes Phylogeny and Classification the uniflagellate, and the genus Euglena is probably paraphyletic rDNA and chloroplast SSU and LSU rDNA (Kim et al., 2015; (Linton et al., 1999). Figure 3). Several subsequent studies were also based on the SSU A recent analysis based on reconstruction of the inferred rDNA gene; however, these were combined with morphological ancestral state revealed many well-supported clades defined by characters used for genus delimitation (Linton et al., 2000; apomorphic morphological characters (e.g., the presence of a Moreira et al., 2001; Müllner et al., 2001; Marin et al., 2003; lorica and mucilaginous stalks), as well as by homoplastic traits Nudelman et al., 2003; Kosmala et al., 2005). Marin et al. (2003) (e.g., rigid cells and the presence of large paramylon grains; provided the first revision of the group in which a diagnosis of Karnkowska et al., 2015). This study also revealed that the Euglenophyceae Schoenichen, 1925 was emended by adding the were lost twice during the evolution of phototrophic nuclear-encoded SSU rRNA molecule with A-U as the first base euglenids, and that mucocysts, which occur only in the genus pair of Helix 39 (Marin et al., 2003). Euglenophyta was recovered Euglena, evolved independently at least twice (Karnkowska et al., as a monophyletic group and was divided into the orders 2015). Eutreptiales and Euglenales. Within this group, Euglenophyceae Multi-gene phylogenetic approaches revealed that all also constituted a monophyletic group, with strong support; phototrophic euglenids form a monophyletic lineage in which whereas Petalomonas cantuscygni and P. trichophorum (both the chloroplast was acquired through secondary endosymbiosis, phagotrophic) and the Distigma clade (formed by saprophytic i.e., a phagotrophic euglenid host engulfed and enslaved a species) showed paraphyletic divergence (Marin et al., 2003). An eukaryotic green alga. Studies based on the chloroplast genome interesting tool used by these authors to separate taxa was the demonstrated that the genus Pyramimonas, a green marine non-homoplasious synapomorphies (NHS), i.e., the characters prasinophyte, is the closest relative of the euglenid chloroplasts without known parallels in other taxa. (Turmel et al., 2009; Hrdá et al., 2012). In addition to these studies are those of Milanowski et al. (2001), who examined the phylogeny of the genus Euglena based Chloroplast Genome on the chloroplast small subunit rDNA (16S rDNA), and others Early vs. late origin of plastids hypothesis on rbcL genes (Thompson et al., 1995), chloroplast ribosomal Although our understanding of the origin of the diversity and large subunit cpLSU rDNA (Kim and Shin, 2008), PAR gene of the evolution of the cpGenomes in euglenids remains unclear, sequences (par1 and par2) (Talke and Preisfeld, 2002), and the most investigators accept the hypothesis of a late plastid to cytosolic form of the heat-shock protein 90 gene (hsp90; Breglia explain the origin of chloroplasts in the group (Hallick et al., 1993; et al., 2007). Vesteg et al., 2010; Bennett et al., 2012; Hrdá et al., 2012). Under this hypothesis, chloroplasts in the Euglenophyceae originated Multi-Gene Phylogenies from a secondary endosymbiosis event with a green alga similar Taxonomic and phylogenetic molecular studies on phototrophic to Pyramimonas, which occurred after the separation of the euglenids based on multi-gene analysis combining different heterotrophic lineage of Peranema, but before the segregation molecular markers with morphological characters have increased of the basal lineages Eutreptiella and Eutreptia (Leander et al., in number. Examples include studies using nuclear SSU and LSU 2001). rDNA (Triemer et al., 2006; Ciugulea et al., 2008); chloroplast SSU rDNA (16S); cytoplasmic SSU rDNA (Milanowski et al., Donor of chloroplast 2006); nuclear SSU, LSU rDNA, and chloroplast SSU rDNA (16S) Comparative analyses between the genomes of the chloroplasts sequences (Linton et al., 2010); cytoplasmic and chloroplast small (cpGenomes) generated new understandings of evolutionary subunit rRNA, and the internal transcribed spacer (ITS2) of questions among the euglenids and with their ancestor, cytoplasmic rDNA (Zakry´s et al., 2002); small-subunit rRNA, similar to Pyramimonas, although the origin of the diversity large-subunit rRNA α-tubulin, β-tubulin, actin, and cytosolic and evolution of the cpGenomes in the group still require heat shock protein 90 sequences (Kim et al., 2006); and α-tubulin, further clarification. Complete cpGenome sequences of euglenids β-tubulin, elongation factor 1α (EF-1α), elongation factor 2 (EF- have been reported for 13 phototrophic species (Sheveleva 2), cytosolic heat shock protein 70 (HSP70), and cytosolic heat and Hallick, 2004; Hrdá et al., 2012; Pombert et al., 2012; shock protein 90 (HSP90; Simpson et al., 2006). Triemer and Wiegert et al., 2012, 2013; Bennett et al., 2014; Bennett and Farmer (2007) published a thorough revision of the systematic Triemer, 2015). Results of these studies revealed an intense and molecular phylogeny of the euglenids described up to the loss of genes occurring in the chloroplast genomes from the year 2006. common ancestor of Pyramimonas parkeae and E. gracilis, to Analysis of nuclear SSU rDNA and plastid SSU and LSU rDNA the present representatives of E. gracilis, as a consequence led to splitting of the photosynthetic euglenids into two major of secondary endosymbiosis (Turmel et al., 2009); and that monophyletic groups, represented by the clades Euglenaceae and these gene losses may have been accompanied by the loss of the newfamilyPhacaceae (Kim et al., 2010). Another study, based photosynthesis capacity, for example in Euglena longa (Gockel on nuclear SSU and LSU rDNA, chloroplast SSU rDNA, and two and Hachtel, 2000; Busse and Preisfeld, 2003). The organization nuclear protein-coding sequences (hsp90 and psbO), indicated of the cpGenome proved to be diversified in the lineages of the monophyly of the families Euglenaceae and Phacaceae E. gracilis and Eutreptiella gymnastica, and also in the lineage (Karnkowska et al., 2015). More recently, the monophyly of both that originated Euglena (Hrdá et al., 2012; Wiegert et al., families was confirmed in a study based on nuclear SSU and LSU 2012).

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FIGURE 3 | Diagram of a Bayesian tree for the phototrophic euglenids based on mutigenes (nSSU, nLSU, cpSSU, hsp90, and psbO). Numbers at internal nodes represent posterior probability (pp) values followed by bootstrap support (bs) values. Support values below 0.75 pp and below 50 for bs are marked with dashes (adapted from Karnkowska et al., 2015).

Origin and evolution of introns However, remains unclear how the increase in the number of An analysis of the cpGenome of Monomorphina aenigmatica introns occurred, and what alterations in their physical form supported the late-intron hypothesis and the occurrence of occurred in the euglenids (Pombert et al., 2012). two distinct and independent acquisitions of introns in the cpGenomes of the euglenids (Pombert et al., 2012). Gene content and synteny among species The identification of six insertion sites, with an intermediate Instances of synteny in the gene contents were identified between state in the appearance of twintrons, led to the supposition the cpGenomes of E. gymnastica vs. E. gracilis, and E. viridis vs. that the first twintron (group III twintron) that appeared in E. gracilis (Hrdá et al., 2012; Wiegert et al., 2012). the cpGenomes of the euglenids is that found in the gene that A more recent analysis of the cpGenomes of Cryptoglena codes for psbC (Pombert et al., 2012). Chloroplast psbC gene was skujai, E. gracilis var. bacillaris, E. viridis, Euglenaria anabaena, also recorded in other phototrophic euglenids, e.g., E. gracilis, E. Monomorphina parapyrum, and Trachelomonas volvocina gymnastica, and Eutreptia viridis (Hrdá et al., 2012; Wiegert et al., showed that these genomes, generally, do not have the very 2012). conserved gene order within the Euglenaceae, and also that The presently available evidence suggests that the introns strains of E. gracilis and E. viridis have marked synteny, found in cpDNAs of euglenids were acquired after the secondary indicating that synteny is a distinctive feature of the taxon endosymbiosis. The presence of a single intron in P. parkeae, (Bennett and Triemer, 2015). A surprising aspect was that unrelated to those found in the DNAs of the chloroplasts different genera (C. skujai, E. anabaena, M. aenigmatica, M. of euglenids, as well as the small number of introns in the parapyrum, and Strombomnas acuminata) share the same cpGenome of E. gymnastica (basal lineage of the euglenids) synteny, a feature that was previously known only among species support the idea that the supposed ancestor of the group was of the same genus (Bennett and Triemer, 2015). Other new poor in introns; and also that the cpGenome of the ancestor and important aspects noted were: (i) unusually high variability already possessed the psbC intron (Doetsch et al., 1998) and among the genomes in the family Euglenaceae, unlike their gene that other introns were acquired later in the course of evolution. contents, which are extremely conservative; (ii) absence of a

Frontiers in Ecology and Evolution | www.frontiersin.org 6 March 2016 | Volume 4 | Article 17 Bicudo and Menezes Phylogeny and Classification pattern of intron numbers in the cpGenome in the Euglenaceae; of the majority of the pre-mRNA in the process depending on and (iii) the presence or absence of twintrons is apparently not spliceosome-trans-splicing (Milanowski et al., 2014). A unique specific to species, but rather, to genera (Bennett and Triemer, aspect was the presence of three types of introns in the nuclear 2015). genome of E. gracilis: (i) conventional, (ii) nonconventional, and (iii) intermediate (Breckenridge et al., 1999; Canaday et al., 2001; Mitochondrial Genomes Vesteg et al., 2010; Milanowski et al., 2014). These introns have Few studies on the mitochondrial genome (mtGenome) in been studied in order to improve understanding of the evolution phototrophic forms have been conducted. Most studies have of the euglenids and their respective genomes, as well as to concentrated on the mitochondrial genome of E. gracilis (Ray and reconstruct the evolution of the gene architecture in eukaryotes Hanawalt, 1965; Crouse et al., 1974; Nass et al., 1974; Tessieretal., (Rogozin et al., 2012). 1997; Yasuhira and Simpson, 1997; Roy et al., 2007). Analysis of the tuba and tubb genes in 20 species of euglenids The mtGenome of E. gracilis comprises numerous DNA revealed different patterns of distribution of conventional and dispersed in different-sized linear fragments, as well as some nonconventional introns. Whereas, the position of conventional circular fragments, two fragments of SSU and two of LSU of the introns is conservative, the nonconventional introns are unique rRNA (Roy et al., 2007; Flegontov and Lukeš, 2012; Moreira et al., to species or groups of closely related species (Milanowski et al., 2012). Moreover, various dispersed fragments of three protein- 2014). coding genes (cox1, cox2, and cox3) have also been identified in Eleven events of losses of conventional introns, compared the mitochondrial genome of E. gracilis (Spencer and Gray, 2011). with 15 other events of acquisition of nonconventional introns, The regions adjacent to the gene fragmentation lack were identified in phototrophic forms by Milanowski et al. the classical organelle intron signature in the E. gracilis (2014). These results suggest that nonconventional introns were mitochondrial DNA (Moreira et al., 2012), and its significant more commonly acquired during the course of their evolution fraction is non-coding and contains many repetitions, than were the conventional introns, and that the two types of interspersed with fragments of a handful of protein-coding introns must have had different origins (Milanowski et al., 2014). genes (Flegontov and Lukeš, 2012). Dobáková et al. (2015) An important aspect to consider in the evolution of the study revealed not only the existence of the cytochrome B gene genome and its components is the process of horizontal gene (gene cob) as well as the presence of nad1, nad4, and nad5 transfer (HGT), interpreted as an important directing force in genes in mt-genome of E. gracilis. Genes encoding subunits of the evolution of many groups of prokaryotes and eukaryotes, ATP synthase were not found in the mt-Genome of E. gracilis as well as their genomes (Keeling and Palmer, 2008). HGTs (Dobáková et al., 2015). also constitute potential sources of information on phylogeny The structure and general organization of the mtGenome and on the adaptive evolution of species (Schönknecht et al., of E. gracilis are extremely close to those observed in the 2014; Katz, 2015). Most of the information on HGTs is on dinoflagellates, whose mtGenome also contains three protein- the prokaryotes, although an increasing number of studies coding genes (cob, cox1, and cox3) in the form of numerous have provided evidence of a concentration of large numbers fragments of various sizes, thus reinforcing the hypothesis of of genes in the eukaryote nuclear genomes that are derived cascades of convergent evolution between the two lineages (Lukeš from organelles, by means of endosymbiotic gene transfer (EGT; et al., 2009; Spencer and Gray, 2011; Flegontov and Lukeš, 2012). Timmis et al., 2004; Schönknecht et al., 2014). The formation According to this hypothesis, although the dinoflagellates and of organelles, accompanied by massive transfers of genes with euglenozoans belong to phylogenetically distinct lineages, they subsequent evolution to the genomes of the host, has transferred share innumerable characters in addition to the mtGenome, complex functional systems, such as photosynthesis, leading to e.g., the presence of a paraxonemal/paraflagellar rod, nucleus important evolutionary adaptations (Schönknecht et al., 2014). with trans-splicing, and a secondary chloroplast with three In phototrophic euglenids, some studies have indicated that HGT surrounding membranes, among others (Lukeš et al., 2009; is a possible directing force of some evolutionary processes. Flegontov and Lukeš, 2012). Analysis of an expressed sequence tag (EST) genetic marker in E. gracilis showed that the nuclear genes in this species have Nuclear Genomes a complex history, and that many aspects of the integration of Approaches based on the nuclear genome and its components the nuclear genome of E. gracilis with the genes of the green have been used to elucidate evolutionary processes among the alga acquired through secondary endosymbiosis remain unclear prokaryotic and eukaryotic lineages, as well as phylogenies (Ahmadinejad et al., 2007). Additionally, molecular phylogenies of different groups of organisms. In general, studies on suggest that genes of the “red lineage” are present in the nuclear nuclear genomes in euglenids are few. Among the phototrophic genome of E. gracilis (Ahmadinejad et al., 2007; Sanchez-Perez euglenids, most studies have dealt with E. gracilis, which even et al., 2008). The presence of 14 genes originating from a red alga, though its genomes has been only partly studied, was found based on analysis of an expanded EST survey using the laterally to have unusual features such as the presence of rRNA genes transferred gene mining pipeline, was detected in E. gracilis and located extrachromosomically, and by thousands of copies of in the heterotrophic P. trichophorum (Maruyama et al., 2011). circular molecules (Cook and Roxby, 1985). The single molecule The 14 phylogenetic trees based on proteins showed, for example, of 28S rRNA was replaced by 13 short RNA molecules (Schnare that the sequence of E. gracilis is monophyletic with the “red ′ and Gray, 1990), and a spice leader is transferred to the 5 -end lineage” and that the homogentisate phytyltransferase (HPT)

Frontiers in Ecology and Evolution | www.frontiersin.org 7 March 2016 | Volume 4 | Article 17 Bicudo and Menezes Phylogeny and Classification protein family of E. gracilis is intimately related to the group of REASSESSMENT OF GENERA AND the Chromalveolata and homologs of red algae (Maruyama et al., SPECIES OF PHOTOTROPHIC EUGLENIDS 2011). These data suggest that the lineage of Euglenida could have undergone a process of cryptic endosymbiosis with an alga According to Leedale (1967), the euglenid flagellates pose a with a red chloroplast, before the present cloroplast from green series of problems for the taxonomist, including duplication algae became established (Maruyama et al., 2011). Possibly, these at the species level, circumscription at the genus level, and genes were acquired through HGT between eukaryotes, resulting assemblage at the order level. It is clear that many of Huber- in the complex mosaic pattern in the genome of euglenids, Pestalozzi’s (1955) 235 species of Euglena are unacceptable suggesting multiple HGTs beginning with the red-alga lineage in duplications, generated when size variations and ecological forms the common ancestor of the euglenids (Maruyama et al., 2011). were described as separate species. For instance, Pringsheim Study of the evolutionary history of the enzymes that (1956), using controlled culture conditions, reduced Huber- participate in the Calvin-Benson cycle in 9 heterotrophic and Pestalozzi’s 235 species to 50 after removing synonyms and 20 phototrophic euglenids indicated that many of these enzymes morphological expressions that were insufficiently described and were probably derived from processes of HGT and/or EGT. unrecognizable. Nuclear-encoded plastid-targeted genes included in the Calvin- Another important aspect raised by Leedale (1967) is the Benson cycle arise from different ancestors derived from multiple absence of sexuality in the euglenid flagellates, and therefore, lineages of Rhodophyta and Chromophyta. Transfer of these the fact that many natural populations are asexual clones, an genes to the nucleus probably preceded the acquisition of the occurrence that may lead to the conclusion that a clone produced green chloroplast in the euglenids (Markunas and Triemer, by a cell with a major morphological peculiarity (acquired by 2015). mutation or a copy error during cell division) can legitimately Szabová et al. (2014) examined the distribution of paralogous be described as a new “species.” Consequently, the species genes encoding the enzymes MAT-MATX (methionine concept in euglenid flagellates remained, in practice, almost adenosyltransferase) in 21 photoautotrophic euglenids and entirely morphological, i.e., species were delimited on the basis two heterotrophic forms, R. viridis and Pyramimonas parkae. of morphology alone. The phylogenetic trees showed that MATX was present only in With the advent of and the recent advances in molecular the phototrophic forms (sub-clade with 70% bootstrap); both phylogeny, taxonomy of the euglenid photoautotrophic forms MAT and MATX in two phototrophic species; and only MAT changed, with proposals of new taxa and combinations. Marin in the heterotrophic euglenids R. viridis and P. parkae. Szabová et al. (2003) is the first taxonomic revision and phylogenetic study et al. (2014) noted that these results, added to the incongruence of Euglenophyceae using molecular data. In that work, among between eukaryotic phylogenies based on MATX and SSU rDNA various taxonomical changes the authors based on nuclear- data, suggest that MATX appeared in the euglenid lineage in a encoded SSU rRNA and SSU rRNA secondary structure redefined single horizontal gene-transfer event (HGT) that occurred after genera, reestablished the genus Monomorphina which is formed the secondary endosymbiotic process of the euglenid chloroplast. by some taxa belonging to traditional genera Phacus and Euglena, and moved a group of species of Euglena with rigid pellicle HOW MANY CLADES ARE THERE WITHIN and many discoid chloroplast without to Lepocinclis EUGLENOPHYCEAE? (Marin et al., 2003). Many other studies continually improved refinement of genera and species concepts in Euglenophyceae. Currently, the Euglenophyceae encompasses three groups, The genus Euglena remains problematic although some progress represented by Rapaza, Eutreptiales and Euglenales (Adl et al., has been made, for example, the analysis of the phylogenetic 2012). Rapaza includes only a single marine species, R. viridis, relationship among species of Euglena and the taxonomy of characterized by mixotrophic cells and a novel feeding apparatus E. viridis, based upon SSU and partial LSU rDNA (Shin and composed of one rod of microtubules, a feeding pocket, and no Triemer, 2004), the description of a new genus, Discoplastis, using vanes (Yamaguchi et al., 2012). SSU and LSU rDNA (Triemer et al., 2006), the reevaluation of The Eutreptiales includes mainly marine and brackish, the taxonomy of species from Euglena with a single axial stellate occasionally freshwater species, in the genera Eutreptia and chloroplast, including the description of two new species based Eutreptiella, that have phototrophic cells with a flexible pellicle on nuclear SSU sequences and morphological data (Kosmala formed by a relatively large number of helical strips, and no et al., 2009), the erection of new genus Euglenaria using conspicuous feeding apparatus (Adl et al., 2012; Yamaguchi et al., combined sequences of nuclear SSU and LSU and of chloroplast 2012). 16S rDNA (Linton et al., 2010). More recently, the taxonomy The Euglenales includes phototrophic and heterotrophic of species from Euglena and Euglenaria was revisited, based species that occur primarily in freshwater environments and have on SSU rDNA and morphology (Karnkowska-Ishikawa et al., a flexible or rigid pellicle, frequently formed by a relatively large 2012, 2013), and Euglena proxima was moved into the new number of helical strips. Phototrophic forms have a pellicle with genus Euglenaformis applying chloroplast genome (Bennett et al., a posterior whorl of short strips, i.e., the strips do not reach 2014). An investigation based upon SSU and LSU nuclear the posterior end of the cell. The Euglenales is divided into two rDNA sequences, combined with morphological data, reinforced monophyletic families, Phacaceae and Euglenacaeae (Kim et al., the separation between Trachelomonas and Strombomonas 2010; Adl et al., 2012). (Ciugulea et al., 2008). Other molecular studies using different

Frontiers in Ecology and Evolution | www.frontiersin.org 8 March 2016 | Volume 4 | Article 17 Bicudo and Menezes Phylogeny and Classification genetic markers (nuclear-encoded genes, chloroplast-encoded from other members of Euglena andisasisterof Euglenaria (Kim gene, and nuclear encoded chloroplast gene) confirmed the et al., 2015). separation of those two genera (Karnkowska et al., 2015; Integrating both morphological features and molecular data Kim et al., 2015). The concept of the genus Lepocinclis was in studies of euglenid evolution is not an easy task, because of broadened by including, among others, Euglena spirogyra, their great plasticity. However, many studies combining both Euglena fusca, Euglena helicoideus, Phacus horridus based onSSU kinds of data have demonstrated that there are many stable and rDNA (Kosmala et al., 2005; Bennett and Triemer, 2012) and characteristic morphological aspects among the phototrophic Cyclidiopsis acus using LSU rDNA (Bennett and Triemer, 2014). lineages, such as cell shape, chloroplast morphology, cell Studies based on combined gene sets also revealed cryptic plasticity, paramylon grain diversity, and the presence of mucus diversity in some euglenid genera. Kim et al. (2013a) separated bodies, mucilaginous stalks, and loricas. Characters such as the the genus Monomorphina into eight species, diagnosed primarily presence of a lorica and mucilaginous stalks (apomorphic), rigid by DNA sequence differences in Helix 17 and the spacer in cells, and the presence of large paramylon grains (homoplasic) Helix 23 of the LSU rRNA gene; five of the species that they correspond to well-defined clades (Karnkowska et al., 2015). recognized were new. Kim et al. (2013b) recognized cryptic The classification of loricate genera and the circumscription of species in Cryptoglena, using a combined data set of nuclear SSU Colacium have always relied heavily on the morphology of the and LSU and plastid SSU and LSU rRNA genes, resulting in the lorica and the mucilaginous stalk, respectively (Brosnan et al., segregation of the genus into five clades, two of which represented 2005; Linton et al., 2010). Lorica ornamentation, ontogeny of the previously known species (Cryptoglena skujai and Cryptoglena lorica development, and pellicle-strip reduction are apomorphies pigra), and the other three were designated as new (Cryptoglena useful in differentiating Trachelomonas from Strombomonas soropigra, Cryptoglena similis, and Cryptoglena longisulca). In (Brosnan et al., 2005), while large paramylon granule types and all strains analyzed, the morphology showed no significant the presence/absence of large granules can be used at the generic species-specific pattern (Kim et al., 2013b). Kim and Shin level to support major clades and generic relationships (Monfils (2014), based on four genes from the cytoplasm and pt-encoded et al., 2011). rDNA, found seven clades defining seven new species of Phacus On the other hand, the characters that are widely used to (Phacus brevisulca, Phacus claviformis, Phacus hordeiformis, circumscribe genera are homoplasic (e.g., lorica shape, presence Phacus longisulca, Phacus minimus, Phacus paraorbicularis, and of a distinctive collar and a tailpiece) and may lead to erroneous Phacus viridioryza). The new species were well-supported as identification of taxa on the basis of the morphogenetic process independent taxa, and showed close relationships with small (Brosnan et al., 2005). Some clades and species of Phacus show Phacus species (Kim and Shin, 2014). congruences between molecular phylogeny and morphological Currently, green euglenids (Euglenophyceae) encompass characteristics such as cell size, presence, or absence of sulcus, the following 14 genera: Ascoglena, Euglena, Euglenaria, and number and shape of paramylon granules (Kim and Shin, Euglenaformis, Eutreptia, Eutreptiella, Discoplastis, Phacus, 2014). Other characters such as location, shape and number Lepocinclis, Monomorphina, Cryptoglena, Colacium, of chloroplasts and pyrenoids, presence of paramylon caps and Strombomonas, and Trachelomonas. mucocysts still need further study in order to form a better Lepocinclis and Discoplastis are circumscribed in the family evaluation of these characters in the identification of species and a Phacaceae, and their monophyly was supported in several studies better understanding of the evolutionary context of euglenids (for (Milanowski et al., 2006; Triemer et al., 2006; Linton et al., more details see Triemer and Farmer, 2007; Linton et al., 2010; 2010; Kim et al., 2015) as was the monophyly of the genera Karnkowska et al., 2015). Monomorphina, Cryptoglena, Colacium, Strombomonas, and Trachelomonas (Kim et al., 2010, 2015; Karnkowska et al., 2015). The genus Phacus was originally recognized as a monophyletic WHAT IS THE CUTTING EDGE OF member of the family Phacaceae by Kim et al. (2010). However, PHOTOTROPHIC EUGLENID more-detailed studies based on multi-gene analyses carried out BIODIVERSITY STUDIES TODAY? by Kim and Shin (2014) and Karnkowska et al. (2015) revealed the paraphyletic nature of the genus. In those two studies, Studies on the phylogenetic relationships and morphological Phacus limnophila and Phacus warszewiczii formed a basal evolution among the phototrophic representatives of euglenids lineage to the Lepocinclis clade. Kim et al. (2015) confirmed have resulted in significant progress, 18 years after molecular the monophyly of Phacus with increasing number of taxa as techniques were first applied to these questions. New genera and suggested by Kim and Shin (2014) and Karnkowska et al. species were described based on single or multigene analyses, (2015). Euglenaformis is positioned as a sister genus to all and new insights on evolution of phototrophic euglenids and other Euglenaceae (Milanowski et al., 2006; Kim et al., 2010; their genomes (plastidial, mitochondrial, and nuclear) have Bennett et al., 2014). Eutreptia and Eutreptiella, both containing provided more evidence about the organization and distribution marine species, correspond to the basal lineage of the euglenid of introns and enzymes across the euglenid lineage, supporting phototrophic clade, thus supporting the hypothesis of a marine the hypothesis that HGTs are an important driving force in the origin of the photosynthetic euglenids (Leander et al., 2001; evolution of the group. Hrdá et al., 2012). The Euglena and Euglenaria clades form a One important aspect is the recent application of culture- paraphyletic lineage, because Euglena archaeoplastidiata diverges independent molecular methods for describing new taxa in

Frontiers in Ecology and Evolution | www.frontiersin.org 9 March 2016 | Volume 4 | Article 17 Bicudo and Menezes Phylogeny and Classification the group (Bennett and Triemer, 2012; Lax and Simpson, from China and Argentina, which limits the construction of 2013; Łukomska-Kowalczyk et al., 2015). Culture-independent a more robust phylogeny for the group. The present body of molecular methods open new prospects for better understanding knowledge lacks material and/or clones from other parts of the of the diversity of microorganisms at the taxonomic or world, particularly the tropics. community level (Giraffa and Neviani, 2001). Since not all The present moment is one of intense reflection worldwide: is organisms can be cultured, the application of methods that it worthwhile to keep on describing new euglenid taxa (genera, are independent of culturing furnishes a truer idea of the species, varieties, and taxonomic formae) on the sole basis of diversity of microorganisms in different environments. Thus, their morphology? How much can molecular-biology data really the application of these methods will be able to give an idea of contribute to the definition of new taxa? Of course, floristic the abundance of euglenid taxa in the different environments, studies must necessarily continue to use the morphological and will surely broaden our understanding of the diversity species concept. There is, at present, no other way to proceed. of the group, not only through new discoveries but also Whereas, a few floras have been published for Asia, Europe and through analyses of little-known and/or rare taxa or groups North America (Northern Hemisphere), for the tropics the only of taxa. published flora is an inventory of the Argentinean phototrophic The application of molecular techniques that do not depend euglenids (Tell and Conforti, 1986). on culturing can also broaden our understanding of the Continuing morphology-based work will undoubtedly biodiversity of the phototrophic euglenids, which is still increase the need for a molecular-biology approach to solve underestimated, mainly due to the paucity of studies carried out those problems that morphology cannot. In short, floristic with tropical material. studies will provide the underpinning for molecular-biology It is essential to expand the present picture by using polyphasic studies. approaches, i.e., integration of different kinds of data such as from morphology, genetics, and ecology, to reach a better AUTHOR CONTRIBUTIONS understanding of the phylogenetic relationships among genera, and of the validity of morphological data for species delimitation All authors listed, have made substantial, direct and intellectual in phototrophic euglenids. contribution to the work, and approved it for publication. The number of available sequences used in molecular-biology publications is still insufficient compared to the total estimated ACKNOWLEDGMENTS number of species in the group. Furthermore, these sequences are almost entirely derived from organisms from Europe, followed We thank the two anonymous reviewers for their constructive by the United States (Northern Hemisphere), and only a few comments and suggestions to the manuscript.

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Eutreptia viridis and Euglena gracilis (Euglenophyta). Protist 163, 832–843. doi: Yubuki, N., Simpson, A. G. B., and Leander, B. S. (2013). 10.1016/j.protis.2012.01.002 Reconstruction of the feeding apparatus in mariagerensis Wiegert, K. E., Bennett, M. S., and Triemer, R. E. (2013). Tracing patterns of provides evidence for character evolution within the Symbiontida chloroplast evolution in euglenoids: contributions from Colacium vesiculosum (Euglenozoa). Eur. J. Protistol. 49, 32–39. doi: 10.1016/j.ejop.2012. and Strombomonas acuminata (Euglenophyta). J. Eukaryot. Microbiol. 60, 07.001 214–221. doi: 10.1111/jeu.12025 Zakry´s, B., Milanowski, R., Empel, J., Borsuk, P., Gromadka, R., and Kwiatowski, J. Yabuki, A., Nakayama, T., Yubuki, N., Hashimoto, T., Ishida, K.-I., and Inagaki, Y. (2002). Two different species of Euglena, E. geniculata and E. myxocylindracea (2011). Tsukubamonas globosa n. gen., n. sp., a novel excavate flagellate possibly (Euglenophyceae), are virtually genetically and morphologically holding a key for the early evolution in “Discoba.” J. Eukaryot. Microbiol. 58, identical. J. Phycol. 38, 1190–1199. doi: 10.1046/j.1529-8817.2002. 319–331. doi: 10.1111/j.1550-7408.2011.00552.x 02020.x Yamaguchi, A., Yubuki, N., and Leander, B. S. (2012). Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy Conflict of Interest Statement: The authors declare that the research was to phototrophy: description of Rapaza viridis n. gen. et n. sp. (Euglenozoa, conducted in the absence of any commercial or financial relationships that could Euglenida). BMC Evol. Biol. 12:29. doi: 10.1186/1471-2148-12-29 be construed as a potential conflict of interest. Yasuhira, S., and Simpson, L. (1997). Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60. Copyright © 2016 Bicudo and Menezes. This is an open-access article distributed J. Mol. Evol. 44, 341–347. doi: 10.1007/PL00006152 under the terms of the Creative Commons Attribution License (CC BY). The use, Yubuki, N., Edgcomb, V. P., Bernhardt, J. M., and Leander, B. S. (2009). distribution or reproduction in other forums is permitted, provided the original Ultrastructure and molecular phylogeny of Calkinsia aureus: cellular identity of author(s) or licensor are credited and that the original publication in this journal a novel clade of deep-sea euglenozoans with epibiotic bacteria. BMC Microbiol. is cited, in accordance with accepted academic practice. No use, distribution or 9:16. doi: 10.1186/1471-2180-9-16 reproduction is permitted which does not comply with these terms.

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GLOSSARY the flagellum/flagella emerge: it varies in shape, size, and ornamentation. 1. ATP synthase (adenosine triphosphate syntetase): Enzyme 16. Mastigonemes: Also termed flagellar hairs, these are fine that catalyzes the synthesis of ATP from adenosine filamentous appendages associated with the flagella in many diphosphate (ADP) and inorganic phosphate (Pi) under flagellates. They are usually arranged in one or more rows aerobic and anaerobic cell growth. of thin, delicate, non-tubular units. In contrast, tubular hairs 2. Bodonids: Include organisms that form an order of consist of two distinct regions, one of which is thick and Kinetoplastea. tubular. In the case of euglenids, mastigonemes are non- 3. Closed mitosis: A type of cell division in which the nuclear tubular. membrane remains intact throughout the mitotic cell cycle. 17. MAT (methionine adenosyltransferase): Is an enzyme that 4. Collar: A sharply defined neck surrounding an apical catalyzes the synthesis of S- adenosylmethionine, which is pore through which the flagellum emerges that occurs in the major methyl donor and used as a substrate in a variety Trachelomonas genus. of methylation reactions. 5. Conventional intron: Formed by five snRNPs (small nuclear 18. MATX: Genes encoding a divergent form of MAT. ribonucleoprotein particles), together with numerous less 19. Metaboly: Euglenoid movements with irregular changes in stably associated proteins; the core of the spliceosome the cell shape through various peristaltic-like motions. is conserved in all well-characterized eukaryotes. The 20. Mixotrophic: Feeding which combine phototrophic and spliceosome interacts with specific sites in the intron and the heterotrophic nutrition modes. flanking exons to ensure accurate and efficient splicing. They 21. MRT: A complex array of microtubules associated with the have canonical intron boundary nucleotides- containing feeding apparatus of some phagotrophic and phototrophic characteristic GT. . . AG boundary sequences flagellates; sometimes understood as a vestigial cytopharynx 6. Cytopharynx: A gullet-like canal or groove between the or vestigial feeding apparatus (Shin et al., 2002). cytostome and the endoplasm, through which food is 22. Mucocystis (= mucus bodies): A kind of ejectile organelle or ingested. extrusome located beneath the cell surface, uniform in size, 7. Cytoskeleton: Network of microtubules and microfilaments spherical or spindle-shaped, with openings located between that provide shape, mechanical resistance to deformation, periplast folds. and aid for cell migration; it also aids intracellular transport 23. NAD (nicotinamide adenine dinucleotide): Ubiquitous and movement. coenzyme that occurrs in all living cells, and functions in 8. Cytostome: The mouth opening into the cytopharynx. oxidizing (NAD+) and reduction reactions (NADH); nad1, 9. Extrusomes: Membrane-bounded extrusible bodies usually nad2, nad3 are NADH subunits. located beneath the pellicle, which are ejected under 24. NHS (non-homoplasious synapomorphies): Characters chemical or mechanical stimulation. without known parallels in any other taxa, “sensu” Marin 10. Flagellar roots: Microtubular or fibrous, sometimes et al. (2003). amorphous structures that originate from a protruding basal 25. Non-conventional intron: Formed by non-canonical and body of the flagellum. variable dinucleotides at the borders and by the presence 11. GAPDH (glyceraldehyde-3-phosphate dehydrogenase): of two inverted repeats that are adjacent to each border This gene encodes a member of the glyceraldehyde-3- of the intron. These repeats would have the capacity for phosphate dehydrogenase protein family. The encoded base pairing and for bringing together both splice sites in protein has been identified as a moonlighting protein, order to constitute a distinct, and probably spliceosome-free based on its ability to perform mechanistically distinct mechanism for splicing. They are not similar to GT...AG functions. The encoded protein was originally identified intron boundary sequences. as a key glycolytic enzyme that converts D-glyceraldehyde 26. Osmotrophic: Heterotrophic nutrition in which feeding is 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate. by absorption or uptake of dissolved organic molecules by 12. Intermediate intron: Characterized by properties of both osmosis. conventional and nonconventional introns (e.g., shows a 27. Paramylon: It is first of all storage material for euglenids “GT” _5_’ _SS dinucleotide and terminal base pairing). formed by polymerization of a large number of β-1, 3 13. Intron: A DNA section of a gene that does not encode glucans. any part of the protein produced by the gene. The intron 28. Pellicle: The cytoskeletal complex of euglenids, is initially transcribed pre-molecule mRNA, but is then consisting of the plasma membrane, proteinaceous strips, eliminated during the RNA maturation process (or splice) microtubules, and tubular cisternae of the endoplasmic before leaving the cell nucleus. Introns exist mainly in reticulum. eukaryotic cells. 29. PFR (paraxonemal rods): Also termed paraxial rods, are 14. Kinetoplast (= kDNA): DNA molecule within a large composed of discrete filaments structured in lattice-like mitochondrion (several copies of the mitochondrial arrays, with three distinct domains: proximal, intermediate genome) attached to the basal bodies. and distal. Each domain is formed by a complex array of 15. Lorica: A mineralized envelope or shell-like protective 25 nm-thick filaments intercrossed by 7-nm filaments at an ◦ outer covering with an apical opening through which angle of 100 .

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30. Phagotrophic: Heterotrophic nutrition in which feeding is by 36. Spliceosome: Formed by five small nuclear RNAs (small engulfement and digestion of other cells or particles. nuclear ribonucleoprotein particles) and more than 100 31. Polycistronic: A kind of messenger RNA that encodes associated proteins that spliced out noncoding introns at the several proteins and is characteristic of many bacterial and initial transcript from DNA to RNA in the eukaryotic cell chloroplast mRNAs. nucleus. 32. Psb: Gene encoding the CP43 protein of photosystem II of 37. Splicing: Process that removes introns and exons during photosynthesis. RNA transcription. Splicing occurs only in eukaryotic cells, 33. Pyrenoids: Usually roughly spherical and highly since the DNA of prokaryotic cells lacks introns. The snRNA refractive bodies associated with a carbon-concentrating or ribozyme is responsible for cleaving the bonds between mechanism (CCM), which is composed primarily of nucleotides. the Calvin-cycle enzyme Ribulose-1,5-Bis-Phosphate 38. Sulcus: Apical groove that occurs in Phacus Carboxylase/Oxygenase (RuBisCo), sometimes surrounded genus. by a starch sheath; in euglenids the sheath is formed by 39. Synteny: Set of orthologous genes that have the same accumulation of paramylon. local organization in different species, i.e., the sequence 34. Rods: Cluster of microtubules and amorphous conservation among chromosome segments of two or more proteinaceous material longitudinally oriented within species. the cell, present in phagotrophic euglenids. Together with 40. Trans-splicing: Mechanism acting by connecting transcripts the vanes, the rods form the feeding structures, giving of otherwise unrelated genes. structural support to the cytostome when engulfing prey 41. Twintron: Special intronic arrangement in which cells. introns of two different types occupy the same gene 35. Spliced leader (SL): Is a gene that generates a functional position. ncRNA that is composed of two regions: an intronic region 42. Vanes: A set of membranous folds that surround the of unknown function (SLi) and an exonic region (SLe), cytostome in phagotrophic euglenids. Vanes originate from ′ which is transferred to the 5 -end of independent transcripts the rods. As the prey is engulfed, the vanes rotate in a yielding mature mRNAs, in a process known as spliced leader pinwheel-like fashion, creating a space within the feeding trans-splicing (SLTS). apparatus.

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